Derivative of Butylphthalide and Preparation Method and Use Thereof

ABSTRACT

(−)-(S)-3-(3′-hydroxy)-butylphthalide (a compound shown by Formula I) and an ester formed of the same and an acid are proved by experiments to be applicable to treatment and prevention of cerebral ischemic diseases and have a sleep-improving function. The acid refers to a pharmaceutically acceptable inorganic or organic acid. The inorganic acid refers to nitric acid, sulfuric acid, or phosphoric acid. In addition to an acid radical, the organic acid at least comprises at least one of an amino group, a hydroxyl group, and a carboxyl group. None of the compound shown by Formula I and the ester thereof is water-soluble. An ester generated from the compound and the acid further react with an acid or a base to generate a salt which is water-soluble and is used to prepare injection preparation. The experiment proves that the salt does not stimulate vessels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/351,424, filed on Apr. 11, 2014, which is a national phase filingunder 35 U.S.C. 371 of International Application No. PCT/CN2012/081963filed on Sep. 26, 2012, which claims the benefit of and priority toChinese Patent Application No. 201110309074.X filed Oct. 13, 2011, eachof which applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention belongs to the medical field of a compound,relates to (−)-(S)-3-(3′-hydroxy)-butylphthalide and an ester formed ofthe same with an acid, and discloses the preparation method and usethereof.

BACKGROUND OF THE INVENTION

Butylphthalide has improving effects on impairment of central nervoussystem function in patients with acute ischemic stroke, can promote therecovery of function in patients, and is mainly converted in vivo intotwo metabolites, 3-3-(3′-hydroxy)butylphthalide and3-hydroxy-3-butylphthalide:

Guangdong Zhongke Drug Research & Development Co. Ltd. and Shandong LvyeNatural Drug Research & Development Co. Ltd. synthesized a succinic acidester, a glycine ester and a phosphoric acid ester of the Metabolite I,and illustrated the use thereof for preventing cerebral ischemicdiseases in 2008. Patent application 200410036628.3 discloses a noveluse of butylphthalide homologs 3-(3′-hydroxy)butylphthalide and3-hydroxy-3-butylphthalide. Both of them are proved to have thefollowing effects:

-   1) significantly improving neurologic symptoms in rats caused by    cerebral ischemia due to brain trauma;-   2) improving memory disorder in rats caused by cerebral ischemia;-   3) relieving cerebral edema in rats caused by cerebral ischemia;-   4) reducing stroke in rats caused by cerebral ischemia;-   5) improving energy metabolism in rats caused by cerebral ischemia;-   6) increasing cerebral blood flow in the ischemic brain region;-   7) reducing the area of cerebral infarction in rats with local    cerebral ischemia and relieving symptoms of neurological deficit;-   8) anti-platelet aggregation and anti-thrombosis;-   9) preventing and treating dementia.

Researchers in our company discover that the Metabolite I has twodifferent optical isomers, and have prepared the compound of3-3-(3′-hydroxy)butylphthalide (Metabolite I) in both S- andR-configurations as is shown in the following figures, by a method ofasymmetric synthesis:

Furthermore, it is found by animal tests that the R-configuration cannotaffect the cerebral infarct volume in rats with cerebral ischemia andimprove the symptoms of cerebral ischemia, while the S-configuration haseffects of improving the symptoms of cerebral ischemia and decreasingthe cerebral infarct volume in rats with cerebral ischemia. It is alsofound that the S-configuration of the Metabolite I (the compound ofclaim 1) has the effect of improving sleep.

The S-configuration of the Metabolite I (the compound of claim 1) is anoily liquid and is insoluble in water. Therefore, in order to prepare itinto a dosage form of injectable solution, we have conducted furtherstudies to allow it to become a water-soluble compound by forming anester with an acid and then forming a salt, to meet the requirement ofthe formulation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a derivative of3-(3′-hydroxy)butylphthalide for ischemic stroke, and the preparationmethod and use thereof.

To achieve the purpose mentioned above, the present invention employsthe following technical solutions.

Preparing the compound of formula 1 as shown in the following figure,

and preparing the compound in R-configuration as shown in the followingfigure,

by asymmetric synthesis.

It is proved by animal tests that the compound in R-configuration doesnot have the effect of reducing the cerebral infarct volume of cerebralischemia in rats, cannot improve the symptoms of cerebral ischemia, anddoes not have the effect of improving sleep, while the S-configurationhas very good effects of improving the symptoms of cerebral ischemia,and can improve the sleeping state in animals.

The compound of formula 1 is an oily substance insoluble in water.Therefore we allow it to form an ester with an acid, wherein the acidrefers to a pharmaceutically acceptable inorganic or organic acid. Theinorganic acid refers to nitric acid, sulfuric acid or phosphorus acid.The organic acid further contains one type of group, and at least onegroup selected from amino, hydroxy or carboxyl group in addition to acidradical. The salt from an ester is soluble in water and can be preparedinto the dosage form of an injectable solution or lyophilized powderinjection.

The organic acid can be amino acids, specifically refer to glycine,alanine, lysine, arginine, serine, phenylalanine, proline, tyrosine,aspartic acid, glutamic acid, histidine, lecine, methionine, threonine,pyroglutamic acid, tryptophan or valine.

Wherein, an ester formed with glycine is preferred, which is as shown inthe following figure:

The organic acid can also be a dicarboxylic acid, specifically camphoricacid, malic acid, citric acid, maleic acid, succinic acid, oxalic acid,glutaric acid, ethanedioic acid or malonic acid.

Wherein, an ester formed with succinic acid is preferred, which is asshown in the following figure:

The organic acid can also refer to pamoic acid, hydroxynaphthoic acid,gentisic acid, salicylic acid, hydroxyacetic acid, mandelic acid, lacticacid, 4-acetamidobenzoic acid or nicotinic acid.

The compound of formula I forms an ester with an inorganic acid,preferably phosphoric acid, which is as shown in the following figure:

The above ester further forms a salt, so as to be prepared into awater-soluble compound to solve the problem of water-solubility, andthus can be used to prepare an injectable dosage form.

An ester formed by the compound of formula I with glycine preferablyforms a hydrochloride, which is as shown in the following figure:

The present invention further provides a salt of the dibasic acid estersof the compound of formula I, which refers to a salt formed withpotassium, sodium, magnesium or organic amine. The organic amine radicalcan be tromethamine, diethanolamine, triethanolamine, glycine, lysine orarginine.

Wherein, the sodium salt is preferred, which is shown in the followingfigure:

In order to solve the problem of water-solubility, the ester formed bythe compound of formula I with phosphoric acid can further form a saltwith physiologically acceptable base, which refers to sodium salt,potassium salt, magnesium salt or organic amine salt. The organic amineincludes lysine, glycine, arginine, tromethamine, diethanolamine ortriethanolamine.

The ester formed by the compound of formula I with phosphoric acidpreferably forms a disodium salt, which has the following structuralformula:

The present invention further provides a pharmaceutical composition fortreating ischemic stroke, characterized in that the compositioncomprises a therapeutic effective amount of the compound of generalformula (1) or a salt thereof and a pharmaceutical acceptable carrier.The pharmaceutical composition can be an oral formulation or aninjectable formulation.

Experiments for muscular and vascular irritations indicate that, it isadministered by injection after the ester formed by the compound offormula I with an acid further forming a salt. No muscular and vascularirritations are observed, so that it can be used as injectableformulation.

DETAILED EMBODIMENTS Example 1 Preparation of Various Compounds

The compounds herein have very strong continuity. Therefore, in order todescribe the preparation method of various compounds in a detailed,accurate and convenient way, the method is illustrated by way of oneexample. In the following synthetic route, each compound is indicated bya serial number. For a more concise illustration, in the preparationmethod below, the compounds are replaced by the serial numbers.

When the R in the above scheme is different groups, different compounds1a, 1b and 1c are obtained.

(1). Synthesis of Compound 3:

Diisopropylamine (1.3 mol) was placed into a 2000 mL round bottom flask,and 1000 mL anhydrous dichloromethane was added. After dissolving,triethylamine (2.0 mol) was added, and Compound 2 (1.0 mol) was addeddropwise in an ice bath. After completing the dropwise addition, thereaction mixture was warmed to room temperature, and stirred overnight.500 mL Dichloromethane was added to dilute, and the reaction mixture waswashed respectively with 5% dilute hydrochloric acid (500 mL×1), water(500 mL×1) and brine (500 mL×1), dried over anhydrous sodium sulfate,and concentrated, to obtain 202.4 g Compound 3. The crude yield was 99%,MS (m/z): 206.1.

(2). Synthesis of Compound 4:

Compound 3 (0.8 mol) was dissolved in 500 mL dry tetrahydrofuran, andtert-butyllithium (1.0 mol) was gradually added dropwise at −78° C.After the completion of the dropwise addition,tetramethylethylenediamine (1.2 mol) was added, and stirred at −78° C.for 30 min. Lactone (1.0 mol) was added dropwise into the above mixture,gradually warmed to room temperature, and continued to react for 5 hr.The reaction was quenched by adding saturated ammonium chloridesolution, and the organic solvent was removed by reduced pressuredistillation. The residue was extracted by ethyl acetate, concentratedunder reduced pressure, and recrystallized, to obtain 222.4 g Compound4. The yield was 91%.

HNMR (400 Hz, CDCl₃): 8.08-8.06 (m, 1H), 8.04-8.02 (m, 1H), 7.63-7.61(m, 1H), 7.60-7.58 (m, 1H), 3.95-3.93 (m, 2H), 3.40-3.38 (m, 1H), 2.55(t, J=1.2 Hz, 2H), 1.64-1.62 (m, 2H), 1.25 (d, J=1.5 Hz, 12H), 1.21 (d,J=1.6 Hz, 3H); MS(m/z): 306.2.

(3). Synthesis of Compound 5:

220 g Compound 4 (0.7 mol) was dissolved in 500 mL methanol, sodiumborohydride (1.40 mol) was added in small batches in an ice bath, andstirred overnight at room temperature under the protection of nitrogengas. After the completion of reaction, about 20 mL concentratedhydrochloric acid was added dropwise to decompose the excess sodiumborohydride. Methanol was removed by reduced pressure distillation. Theresidue was extracted by dichloromethane, washed with water, washed withbrine, dried over anhydrous sodium sulfate, concentrated, andrecrystallized, to obtain 204.8 g Compound 5. The yield was 95%.MS(m/z): 308.2.

(4). Synthesis of Compound 6:

Compound 5 (0.60 mol) was dissolved in 500 mL toluene, catalytic amountof p-toluenesulfonic acid (about 1%) was added, and boiled slightly andrefluxed under the protection of nitrogen gas for 8 days. Toluene wasremoved by reduced pressure distillation, dichloromethane was added todilute, and the residue was washed with water, washed with brine, driedover anhydrous magnesium sulfate, and passed through a column, to obtain106.4 g Compound 6. The yield was 86%. MS(m/z): 207.2.

(5). Synthesis of Compound 7:

Compound 6 (0.2 mol) was dissolved in 200 mL methanol, sodium hydroxideaqueous solution (16 g/40 mL) was added under stirring, and completedover 15 min. The system was heated to become a homogenous solution, andcontinued to react at room temperature for 2 hr. The reaction wasquenched, and methanol was removed by reduced pressure distillation. Anappropriate amount of distilled water was added into the residualsolution to dilute. The system was cooled to −5° C. or below, andadjusted to pH=3˜4 with 5% hydrochloric acid solution, and extractedwith diethyl ether (100×3). The diethyl ether extracts were pooled, andthe solution was cooled to −5° C. or below. 0.2 mol(+)-(R)-α-Phenylethylamine was slowly added dropwise. The system wasmaintained at a temperature of −5° C. or below, and allowed to stand for3 hr. Plenty of crystals were precipitated, and the system was filteredto collect the crystals. The crystals were recrystallized twice withacetone or ethyl acetate to obtain 20.3 g crystals, and theconcentration of the crystals was 15 g crystals/100 mL solvent. Thecrystals were dissolved in 10 volumes of distilled water, and sodiumhydroxide was added to adjust the solution to pH=13.(+)-(R)-α-Phenylethylamine was recovered by extraction with diethylether, and the aqueous phase was adjusted to pH=2 with hydrochloricacid, extracted with diethyl ether, dried, and concentrated, to obtain acrude product of (+)-(R)-3-(3′-hydroxy)butylphthalide. The crude productwas recrystallized with ethanol to obtain 8.7 g Compound 7. The yieldwas 21%. [α]_(D)=+66.80 (c=1.02, CH₃OH).

HNMR (400 Hz, CDCl₃): 7.91-7.89 (m, 1H), 7.41-7.39 (m, 1H), 7.33-7.31(m, 1H), 7.30-7.28 (m, 1H), 5.24 (t, J=1.2 Hz, 1H), 3.40-3.38 (m, 1H),2.55 (t, J=1.2 Hz, 2H), 2.05-2.03 (m, 2H), 1.45-1.43 (m, 2H), 1.21 (d,J=1.6 Hz, 3H); MS(m/z): 207.2.

(6). Synthesis of Compound 8:

Compound 6 (0.2 mol) was dissolved in 200 mL methanol, sodium hydroxideaqueous solution (16 g/40 mL) was added under stirring, and completedover 15 min. The system was heated to become a homogenous solution, andcontinued to react at room temperature for 2 hr. The reaction wasquenched, and methanol was removed by reduced pressure distillation. Anappropriate amount of distilled water was added into the residualsolution to dilute. The system was cooled to −5° C. or below, andadjusted to pH=3˜4 with 5% hydrochloric acid solution under stirring,and extracted with diethyl ether (100×3). The diethyl ether extractswere pooled, and the solution was cooled to −5° C. or below. 0.2 mol(S)-α-phenylethylamine was slowly added dropwise. The system wasmaintained at a temperature of −5° C. or below, and allowed to stand for3 hr. Plenty of crystals were precipitated, and the system was filteredto collect the crystals. The crystals were recrystallized twice withacetone or ethyl acetate to obtain 20.3 g crystals, and theconcentration of the crystals was 15 g crystals/100 mL solvent. Thecrystals were dissolved in 10 volumes of distilled water, and sodiumhydroxide was added to adjust the solution to pH=13.(S)-α-phenylethylamine was recovered by extraction with diethyl ether,and the aqueous phase was adjusted to pH=2 with hydrochloric acid,extracted with diethyl ether, dried, and concentrated, to obtain a crudeproduct of (S)-3-(3′-hydroxy)butylphthalide. The crude product wasrecrystallized to obtain 8.7 g Compound 7. The yield was 21%.[α]_(D)=−66.80 (c=1.02, CH₃OH).

HNMR (400 Hz, CDCl₃): 7.91-7.89 (m, 1H), 7.41-7.39 (m, 1H), 7.33-7.31(m, 1H), 7.30-7.28 (m, 1H), 5.24 (t, J=1.2 Hz, 1H), 3.40-3.38 (m, 1H),2.55 (t, J=1.2 Hz, 2H), 2.05-2.03 (m, 2H), 1.45-1.43 (m, 2H), 1.21 (d,J=1.6 Hz, 3H); MS(m/z): 207.2.

(7). Synthesis of Compound 1a:

10.0 mmol Compound 8, 10.0 mmol butanedioic anhydride, and 10 mmol DMAPwere dissolved in 50 mL DMF, heated to 85° C., and reacted for 6 hr. Thereaction was quenched, and the 00 was poured into 200 mL ice water,adjusted to pH=2˜3 with 0.1 N hydrochloric acid, and extracted withethyl acetate (100 mL×3). The organic phase was pooled, washed withbrine for three times, dried over anhydrous sodium sulfate, distilledunder reduced pressure, to obtain (S)-3-(3′-succinateester)butylphthalide, which was recrystallized to obtain 2.8 g whitesolid powders. The yield was 91%.

HNMR (400 Hz, CDCl₃): 7.8-7.78 (m, 1H), 7.65-7.63 (m, 1H), 7.49-7.47 (m,1H), 7.44-7.42 (m, 1H), 5.50-5.46 (m, 1H), 3.82-3.74 (m, 1H), 2.72-2.66(m, 2H), 2.62-2.56 (m, 2H), 2.05-2.03 (m, 2H), 1.45-1.43 (m, 2H), 1.21(d, J=1.6 Hz, 3H); MS(m/z): 307.3.

The above (S)-3-(3′-succinate ester)butylphthalide was dissolved in 50mL methanol and 4 mL 10% sodium hydroxide solution, heated to reflux for2 hr, and concentrated to obtain a sodium salt of (S)-3-(3′-succinateester)butylphthalide (Compound 1a), MS(m/z): 305.3.

(8). Synthesis of Compound 1b:

10.0 mmol Compound 8 was dissolved in 50 mL ethyl acetate, and 1.0 mLpyridine and 10 mmol DMAP were added. The mixture was cooled to 0˜5° C.,and 10.0 mmol glycine was added under stirring, and reacted for 6 hr ata temperature maintained at 0˜5° C. The reaction was quenched, and thereaction solution was poured into 100 mL ice water, adjusted to pH=7.0with 0.1 N hydrochloric acid, and allowed to stand to separate theorganic layer. The aqueous phase was extracted with ethyl acetate (50mL×3). The organic phase was pooled, and washed with brine for 3 times,dried over anhydrous sodium sulfate, distilled under reduced pressure toobtain (S)-3-(3′-glycinate ester)butylphthalide, which wasrecrystallized from ethanol to obtain 2.3 g white solid powder. Theyield was 87%.

HNMR (400 Hz, CDCl₃): 7.8-7.78 (m, 1H), 7.65-7.63 (m, 1H), 7.49-7.47 (m,1H), 7.44-7.42 (m, 1H), 5.50-5.46 (m, 1H), 3.82-3.74 (m, 1H), 3.62 (s,2H), 2.05-2.03 (m, 2H), 1.45-1.43 (m, 2H), 1.21 (d, J=1.6 Hz, 3H);MS(m/z): 264.3.

The above (S)-3-(3′-glycinate ester)butylphthalide was dissolved in 50mL acetone and 10 mL diethyl ether, and a diethyl ether solution ofhydrochloride was added dropwise to adjust pH=2. Plenty of white solidwas precipitated, filtered, dried to obtain 2.4 g (S)-3-(3′-glycinateester)butylphthalide hydrochloride (Compound 1b). The yield was 80%.MS(m/z): 264.3.

(9). Synthesis of Compound 1c:

20.0 mL pyridine was added into 100 mL DMF, cooled to −10° C., and 25.0mL phosphorus oxychloride was added under stirring. The mixture wasstirred for 30 min, and then 10.0 mmol Compound 8 was added batchwise.After stirring for 3 hr, the reaction was quenched. The reactionsolution was poured into 100 mL ice water, adjusted to pH=2 with 0.1 Nhydrochloric acid, extracted with ethyl acetate (50 mL×3). The organicphase was pooled, washed with brine for 3 times, and dried overanhydrous sodium sulfate. The solvent was removed by reduced pressuredistillation to obtain (S)-3-(3′-phosphate ester)butylphthalide. Then itwas dissolved in 20 mL ethanol, and 10.6 sodium carbonate was added toreact for 2 hr under a temperature of 30° C. The reaction was quenched,and 100 mL acetone was added. The mixture were allowed to stand andprecipitated at 5° C., filtered, dried under vacuum, to obtain 2.8 g(S)-3-(3′-phosphate ester)butylphthalide disodium salt (Compound 1c),which was a while solid powder. The yield was 85%.

HNMR (400 Hz, CDCl₃): 7.91-7.89 (m, 1H), 7.41-7.39 (m, 1 H), 7.33-7.31(m, 1H), 7.30-7.28 (m, 1H), 5.24 (t, J=1.2 Hz, 1H), 3.40-3.38 (m, 1H),2.05-2.03 (m, 2H), 1.65-1.63 (m, 2H), 1.21 (d, J=1.6 Hz, 3H); MS(m/z):331.2.

Example 2 Preparation of Injectable (−)-(S)-3-(3′-phosphate esterdisodium)butylphthalide (Compound 1c in Example 1) Lyophilized Powder

10 g the prepared (S)-3-(3′-phosphate ester)butylphthalide sodium salt(Compound 1c) was taken, and 1000 ml water for injection was added todissolve, and 60 g mannitol was further added. After dissolvingcompletely, water for injection was additionally added to 1200 ml. Afterdecarburization by activated charcoal, the mixture was filtered throughmicroporous membrane, aliquoted into 7 ml penicillin bottle with 3 mlfor each bottle, plugged, lyophilized, and glanded, to obtain theinjectable lyophilized powder. Specification: 20 mg/bottle.

Example 3 Preparation of Capsules of (S)-3-(3′-phosphate esterdisodium)butylphthalide (Compound 1C)

Formula: (S)-3-(3′-phosphate ester)- 60 g butylphthalide sodium saltlactose 105 g 1000 capsules

(S)-3-(3′-phosphate ester)butylphthalide sodium salt was accuratelyweighed according to the amount in the formula, sieved through 100 meshscreen, and lactose according to the formula amount, which was dried at80° C. and sieved through 80 mesh screen, was added, mixed evenly. Themixture was detected for the contents, and filled into 1 # capsuleshells if qualified, to obtain the capsules.

Example 4 Preparation of Injectable Solution of (S)-3-(3′-phosphateester disodium)butylphthalide (Compound 1c)

Formula: (S)-3-(3′-phosphate ester)- 50 g butylphthalide sodium saltwater for injection 4000 ml 1000 ampoules

(S)-3-(3′-phosphate ester)butylphthalide sodium salt (Compound 1c) wasaccurately weighed according to the amount in the formula, and anappropriate amount of water for injection was added. The pH was adjustedto 6.5-7.2, and water for injection was added to 4000 ml. 2 g activatedcharcoal for injection was added, boiled for 15 min, decarburizedthrough filtering by suction. The solution was filtered through 0.22 μmmicroporous membrane, filled and sealed in glass ampoules, andautoclaved at 115° C. for 30 min, to obtain the injectable solution.

Example 5 Effects on Volumes of Cerebral Infarction in Rats with LocalCerebral Infarction (1) Experimental Materials and Method

Wistar rats, body weight 250˜280 g, were raised separately before andafter the surgical operation, and kept at room temperature of 23˜25° C.All the rats were allowed ad libitum access to food and water. tMCAOmodels were prepared according to the method of longa, et al. Rats wereanaesthetized with 10% chloral hydrate (350 mg/kg, i.p.), and the bodytemperature was maintained at 37±0.5° C. The rat was fixed in supineposition onto an operating table. The skin was cut along the midline ofthe neck, and the common carotid artery (CCA), external carotid artery(ECA) and internal carotid artery (ICA) on the right side were carefullyisolated. The ECA was ligated and cut, and straightened in line with theICA. A small incision was cut on the ECA, and a 4.0 cm long round-headsilicified nylon thread with a diameter of 0.26 mm (coated with 0.1%polylysine) was inserted through this incision into the ICA for about1.85˜2.00 cm, until the starting place of the anterior cerebral arteryof the rat, to block the blood supply of the middle cerebral artery.After 2 hr of ischemia, the nylon thread was pulled out carefully, theECA incision was ligated, and the operational incision was sutured. Theanimal was returned to the cage for 24 hr reperfusion.

(2) Experimental Groups and Administration

Rats were randomly divided into 12 groups: model control group, waterfor injection (100 mg/kg), the administration group of the Compound 7 inExample 1 (25, 50, 100 mg/kg), the administration group of the Compound8 in Example 1 (25, 50, 100 mg/kg), the administration group ofDL-3-(3′-hydroxy)-butylphthalide (DL for short) (25, 50, 100 mg/kg).They were orally administered 10 min after the ischemia caused by MCAblockage.

(3) Determination of the Volume of Cerebral Infarction

After reperfusion injury in the rat for 24 hr, the rat was beheaded andthe brain was taken out immediately. The olfactory tract, the cerebellumand the low brain stem were removed. The brain was coronally cut into 6slices (the first slice to the fifth slice was 2 mm/slice, and the sixthslice was 4 mm), and they were rapidly placed into 5 ml solutioncontaining 1.5 ml 4% TTC and 0.1 ml 1 M K₂HPO₄ to stain (37° C.,shielded from light) for 20˜30 min, wherein they are flipped once every5 min. After the TTC staining, the normal tissue was dark stained andappeared red, and the infarcted tissue appeared white. Each group ofbrain slices were neatly arranged and were taken photos forpreservation. The infarction area in each slice was calculated, and theinfarction volume was finally converted by superposition. The infarctionvolume was expressed in percentage in a cerebral hemisphere, toeliminate the effects of cerebral edema.

Brain infarction volume (%)=(volume of the contralateral hemisphere tothe operated side−volume of uninfarcted portion of the operatedhemisphere)/volume of the contralateral hemisphere to the operatedside*100%

(4) Experimental Results

After 2 hr of ischemia and 24 hr of reperfusion, the volume of cerebralinfarction of the solvent control group was 33.8%. The sham operationgroup did not have any cerebral infarction. The results of the volume ofcerebral infarction in other groups are as shown in Table 1.

TABLE 1 Effects of gavage administration on the volume of cerebralinfarction in rats with local ischemia Volume of Volume of Volume ofcerebral cerebral cerebral infarction infarction infarction (25 mg/kg(50 mg/kg (100 mg/kg Sample group) group) group) Compound 7 19.8% 18.9%18.7.0%   Compound 8 14.6% 13.8% 11.8% DL 17.2% 16.4% 14.5% Controlgroup 20.1%

In comparison with the solvent control group, oral administration ingroups of Compound 8 and DL-3-(3′-hydroxy)-butylphthalide cansignificantly reduce the volume of cerebral infarction, and groups ofCompound 7 did not effectively reduce the volume of cerebral infarction.Groups of (−)-3-(3′-hydroxy)-butylphthalide (i.e. Compound 8) aresuperior to DL-3-(3′-hydroxy)-butylphthalide, indicating that Compound 8in S configuration was the effective active ingredient, while Compound 7in R configuration was not found to have the effect of reducing thevolume of cerebral infarction caused by cerebral ischemia.

Example 12 Effects of Injection Administration of 1a, 1b, 1c on theVolume of Cerebral Infarction (1) Experimental Groups and Administration

Rats of the ischemia blocking model in Example 11 were taken, andrandomly divided into 12 groups: sham operation group, model controlgroup, (water for injection, 10 mg/kg), 1a group (2.5 mg/kg, 5.0 mg/kg,10 mg/kg), 1b group (2.5 mg/kg, 5.0 mg/kg, 10 mg/kg), and 1c group (2.5mg/kg, 5.0 mg/kg, 10 mg/kg). 10 min after ischemia caused by MCAblockage, they were administered intravenously.

(2) Results and Discussion

The determination of the volume of cerebral infarction was the same toExample 4. After 2 hr of ischemia and 24 hr of reperfusion, the volumeof cerebral infarction of the solvent control group was 33.6%. The shamoperation group did not have any cerebral infarction. In comparison withthe solvent control group, each of the groups can significantly reducethe volume of the cerebral infarction. In comparison with the solventcontrol group, all the groups of samples can significantly reduce thevolume of the cerebral infarction, as is shown in Table 2.

TABLE 2 Effects of intravenous administration of the salt of MI ester onthe volume of cerebral infarction in rats with local ischemia Volume ofcerebral Volume of cerebral Volume of cerebral infarction infarctioninfarction Sample (2.5 mg/kg group) (5.0 mg/kg group) (10.0 mg/kg group)1a   21% 15.4% 11.8% 1b 21.3% 16.0% 12.0% 1c 20.6% 15.8% 11.7% Control33.6% group

Example 13 Experiments for Vascular Irritation of 1a, 1b and 1c 1)Design of Experiments

1a, 1b and 1c were taken and dissolved in water for injection, and wererespectively prepared into two concentration groups. The highconcentration group: 4.2 mg/ml, and the low concentration group: 1.4mg/ml. They were administered intravenously through rabbit auricularveins, and the dosage was 5 ml.

2) Administration Method:

8 Healthy New Zealand rabbits were selected, and the test drug in highconcentration and low concentration was respectively injected into theauricular vein of the left ear of the rabbits, while equal volume of aninjectable solution of sodium chloride was injected into the auricularvein of the right ear of the rabbits. The 8 rabbits were administeredsuccessively with the test drug in high concentration and lowconcentration, and then administered respectively with 0.9% injectablesolution of sodium chloride. This is conducted once every day for 3consecutive days. The rabbits were weighed respectively before theadministration, and 48 hr and 14 days after the administration.

3) General Observations and Sampling in Animals

The reactions of the animals and the injection site of the blood vesselwere observed and recorded before the administration every day. 48 hrafter the last administration, 2 New Zealand rabbits administeredrespectively with high concentration and low concentration were executedby bloodletting. The reactions of the blood vessel tissues were observedvisually and recorded, then both ears of the rabbits were cut off fromthe roots of ear (firstly the left ear, then the right ear, and labelthem). Then, a section of specimen of the rabbit ear was cut outrespectively, and fixed in 10% solution of neutral formaldehyde (thespecimen was 8 cm long and 1 cm wide; the incision at the distal end tothe heart was about 0.5 cm away from the first pinprick, and theincision at the proximal end to the heart was about 2 cm away from thethird pinprick, and the proximal end to the heart was the end forhanging). 2 animals administered respectively with the test drug in highconcentration and low concentration were left for further observationuntil the 14^(th) day after the last administration, and pathologicalexamination was conducted.

From the border of the first pinprick, one section was cut at the distalend; and from the border of the third pinprick, two sections were cut atthe proximal end; the blood vessel was cut transversely when preparingthe slices. The preparation of slices was conducted with conventionalparaffin, and the thickness of the slice was 4˜5 μm. H-E staining wasconducted before performing histopathologic examination.

5) Result Judgment

Comprehensive judgment was conducted according to the visual observationand pathological examination. The reactions of the injection site of theanimal blood vessel were visually observed and recorded beforeadministration every day. During the administration, it was visuallyobserved that the inner side and outer side of the vascular epidermis atthe injection site of the rabbit ear in the administration side and thecontrol side in some of the animals administered with the test drug inhigh concentration and low concentration appeared red, with an area from0.1 cm×0.2 cm to 0.2 cm×1.0 cm. 48 hr after the last administration, thevascular profile of the blood vessel of the rabbit ear in both side of 4rabbits administered with the test drug in high concentration and lowconcentration was clear, and the thickness of the rabbit ear wasuniform, and no significant change was observed. 14 days after the lastadministration, necropsy was conducted in 4 rabbits administered withthe test drug in high concentration and low concentration. The vascularprofile of the rabbit ear in both sides was relatively clear, and thethickness of the rabbit ear was uniform, and no significant change wasobserved.

Necropsy in 4 rabbits administered with the test drugs in highconcentration and low concentration was conducted 40 hr after the lastadministration, and the necropsy in the other 4 rabbits administeredwith the test drugs in high concentration and low concentration wasconducted at the end of 2 weeks of recovery period. No significantirritation reaction such as degeneration and necrosis in vasculartissues was observed in all the histopathologic examinations.

Example 13 Effects of Compound 7, Compound 8 andDL-3-(3′-hydroxy)-butylphthalide on Rat Sleep Experiments for ImprovingSleep

Properties of the Samples: the contents in the capsule formulation ofCompound 7 and Compound 8 of the present invention are brown particles.

Source of Animals: Kunming mice, 18˜22 g, male, were clean grade animalsprovided by Guangdong Medical Lab Animal Center. In the breeding room ofthe experimental animals, the temperature was 22±2 ° C., the relativehumidity was 55˜70%, and the animal feed was provided by GuangdongMedical Lab Animal Center.

3 groups of Compound 7, Compound 8, DL-3-(3′-hydroxy)-butylphthalide (DLfor short) were set in the experiment, 25 mg/kg respectively. Anddistilled water control group was additionally set.

Sample Processing: 25 mg respective sample was taken, and added withdistilled water to 20 ml, to form a uniform suspension, for use in thetest.

Route for Administration: gavage

Experimental Method: Hypnosis Test of Pentobarbital Sodium in DosageAbove Threshold:

40 male mice with a body weight of 18˜22 g were selected, and randomlydivided into 4 groups, 10 mice in each group. They were consecutivelyadministered with samples for 30 days, and 15 min after theadministration of the samples by gavage on the 30^(th) day, each groupof animals were i.p. injected with 50 mg/kg.b.w pentobarbital sodium,and the injection amount was 0.2 ml/20 g.b.w. The criterion for judgingfalling asleep was that the righting reflex in a mouse disappeared for 1min or more. The time for falling asleep and the sleeping time of eachgroup of animals within 60 min were observed after administration ofpentobarbital sodium.

Results: Effects of Samples on the Body Weight of Animals

Body weight (g) Number of Beginning of Interim of the End of Groupanimals the test test the test Control group 20 20 ± 1.4 27 ± 1.6 35 ±2.0 Group of 20 20 ± 1.5 27 ± 1.3 35 ± 1.6 Compoud 7 Group of 20 20 ±1.3 27 ± 1.6 35 ± 2.3 Compoud 8 Group of DL 20 20 ± 1.2 27 ± 1.2 35 ±2.4

As is shown in the above table, in comparison with the control group,the body weights of animals in each dosage group have no significantdifference.

Effects on Sleeping Time in Mice Induced by Pentobarbital Sodium inDosage Above Threshold

Number of Time for falling Sleeping time Dosage group animals asleep(min) (min) Control group 10 5.79 ± 2.05 40.96 ± 8.16 Group of 10 5.78 ±1.88 41.16 ± 7.80 Compound 7 Group of 10  3.91 ± 0.99*  53.28 ± 11.12*Compound 8 Group of DL 10 4.68 ± 1.35 45.48 ± 11.23 *P < 0.05, ascompared to the control group (by ANOVA)

As is shown in the above table, in comparison with the control group,the time for falling asleep and the sleeping time of the animals inducedby pentobarbital sodium in dosage above threshold in the group ofCompound 8 ((−)-(S)-3-(3′-hydroxy)-butylphthalide) have significantdifference.

Hypnosis Test of Pentobarbital Sodium in Dosage Below Threshold:

40 male mice with a body weight of 18˜22 g were selected, and randomlydivided into 4 groups, 10 mice in each group. They were consecutivelyadministered with samples for 28 days, and 15 min after theadministration of the samples by gavage on the 30^(th) day, each groupof animals were i.p. injected with 30 mg/kg.b.w pentobarbital sodium,and the injection amount was 0.2 ml/20 g.b.w. The criterion for judgingfalling asleep was that the righting reflex in a mouse disappeared for 1min or more. The time for the occurrence of sleep of each group ofanimals within 25 min were observed after administration ofpentobarbital sodium.

Results: Effects on the Incidence of Sleep in Mice Induced byPentobarbital Sodium in Dosage Below Threshold

Number of Number of animals Incidence of sleep Dosage group animalsfalling asleep (%) Control group 10 2 20 Group of 10 3 30 Compound 7Group of 10  8*  80* Compound 8 Group of DL 10 5 50 *P < 0.05, ascompared to the control group (by chi-square test)

As is shown in the above table, in comparison with the control group,the number of animal falling asleep and the incidence of sleep in theanimals induced by pentobarbital sodium in dosage below threshold in thegroups of Compound 8 ((−)-(S)-3-(3′-hydroxy)-butylphthalide) and DL havesignificant difference.

Summary: after oral administration to mice with the samples for 30 days,the groups of Compound 8 ((−)-(S)-3-(3′-hydroxy)-butylphthalide) and DLhave effects of improving sleep. The effect of the S configuration isgreater than that of the racemic DL configuration, and that of the DLconfiguration is greater than that of the R configuration.

1. A compound for preventing or treating cerebral ischemic disease, thecompound having formula I below:

or having the chemical name: (−)-(S)-3-(3′-hydroxy)-butylphthalide. 2.An ester obtained by reacting the compound according to claim 1 with anacid, said acid selected from a pharmaceutically acceptable inorganicacid or organic acid.
 3. The ester according to claim 2, wherein theinorganic acid is selected from the group consisting of nitric acid,sulfuric acid or phosphoric acid; the organic acid is selected from thegroup consisting of amino acid: glycine, alanine, lysine, arginine,serine, phenylalanine, proline, tyrosine, aspartic acid, glutamic acid,histidine, lecine, methionine, threonine, pyroglutamic acid, tryptophanor valine; dibasic acid: camphoric acid, malic acid, citric acid, maleicacid, succinic acid, oxalic acid, glutaric acid, ethanedioic acid,lactic acid or malonic acid; pamoic acid, hydroxynaphthoic acid,gentisic acid, salicylic acid, hydroxyacetic acid, mandelic acid, lacticacid, 4-acetamidobenzoic acid or nicotinic acid.
 4. The ester accordingto claim 2, wherein the acid is glycine, succinic acid, phosphoric acid,and wherein the ester has formula II, III or IV below:


5. The ester according to claim 2, which is in the form of salt, whereinthe ester in the form of salt is obtained by reacting the compound offormula I with an amino acid to obtain an ester, which is furtherreacted with sulfuric acid, phosphoric acid, sulphonic acid orhydrochloric acid; or obtained by reacting the compound of formula Iwith a dibasic acid to obtain an ester, which is further reacted withsodium, potassium, magnesium, tromethamine, diethanolamine,triethanolamine, glycine, lysine or arginine; or obtained by reactingthe compound of formula 1 with phosphoric acid to obtain an ester, whichis further reacted with sodium, potassium, magnesium, lysine, glycine,arginine, tromethamine, diethanolamine or triethanolamine.
 6. The esterin the form of salt according to claim 5, having formula V, VI or VIIbelow:


7. A method of treating cerebral ischemic diseases comprisingadministrating an effective amount of the compound according to claim 1to a subject in need thereof.
 8. A method of improving sleep comprisingadministrating an effective amount of the compound according to claim 1to a subject in need thereof.
 9. A method of treating cerebral ischemicdiseases and improving sleep comprising administrating an effectiveamount of the ester according to claim 2 to a subject in need thereof.10. A method of treating cerebral ischemic diseases and improving sleepcomprising administrating an effective amount of the ester according toclaim 4 to a subject in need thereof.
 11. A pharmaceutical composition,comprising an effective amount of the compound according to claim 1, anda pharmaceutically acceptable carrier.
 12. A pharmaceutical composition,comprising an effective amount of the ester according to claim 2, and apharmaceutically acceptable carrier.